There is a continuing need in medical practice and research, and in analytical and diagnostic procedures for rapid and accurate determinations of chemical and biological substances which are present in various fluids, such as biological fluids. For example, the presence and quantity of protein must be determined rapidly and accurately for effective research, diagnosis and treatment of many human diseases.
A wide variety of analytical methods have been developed in recent decades to detect the noted substances. The methods have become highly reliable and in some instances, suitable for automation, as well as suitable for use in kit form.
Protein assays have been traditionally carried out in solution, or in test devices where fluids are removed in some fashion from the reagents participating in the assay. Although solution techniques have enjoyed broad acceptance in this area, they typically require analyzer equipment often having intricate solution handling and transport capabilities. Moreover, the analytical equipment used in such assays can involve complex liquid handling, and may require skilled personnel, both for operation and the precise cleaning that may be needed to avoid sample to sample contamination.
An alternative to solution chemistry is the use of dry analytical elements. It should be understood that not all solution-based analytical assays can be adapted for use in dry analytical elements because of interference from coating agents,such as binders,surfactants, and other reagents necessary to promote or facilitate material deposition, sample wetting, and maintain structural integrity of said elements. Moreover, dry analytical elements must use in situ compartmentalization to segregate incompatible components. Such is not the case in solution chemistry where separate liquid storage and successive liquid additions can be employed.
Dry analytical elements and their use are described in numerous publications, including U.S. Pat. No. 4,132,528, U.S. Pat. No. 4,786,605, U.S. Pat. No. 3,992,158, U.S. Pat. No. 4,258,001 to Pierce et al., U.S. Pat. No. 4,670,381 to Frickey et al., WO 82/2601 (published Aug. 5, 1982), European Patent Application No. 051 183 (published May 12, 1982) and European Patent Application No. 066 648 (published Dec. 15, 1982). The entire contents of the noted citations are incorporated herein by reference.
A useful diagnostic indicator for assessing and monitoring patient kidney function is the total protein concentration present in urine. The nature and amount of protein present in urine is varied and dependent on the particular disease state which results in the failure of the kidney to prevent passage of proteins into the urine.
Examples of proteins that may be found in urine include, but are not limited to, albumin, intact immunoglobulins, kappa free-chains, lambda free-chains, retinol binding protein, alpha-l-microglobulin, and beta-l-microglobulin. These proteins differ widely in amino acid composition and molecular weight. The information of interest to the clinician using a urine total protein screening assay is the total mass of protein per unit volume of the urine specimen. The diagnostic assay, that is, the measured signal ideally, should be independent of the nature or type of proteins that may be present. For example, 50 mg/dL of albumin should produce the same signal as 50 mg/dL of any other protein. The normal protein concentration range in human urine is between approximately 5 to 100 mg/dL.
Various means have been used to determine the total protein concentration in biological materials. In many analytical assays, an optical signal, such as absorption in the visible or ultraviolet region of the spectrum, is measured which is proportional to the concentration of protein in the sample. Generally, however, the signal is undesirably a function of the nature of the protein in the sample and not simply related to the mass of protein present. These methods usually rely on one of the following means to generate a detectable optical signal:
1. A complex of protein and Cu(II), the biuret reaction, results in a detectable but small absorption in the visible region of the spectrum.
2. Absorption in the ultraviolet region of the spectrum by aromatic amino acids of the protein may be measured.
3. The amount of protein may be determined by derivitization of specific amino acids with molecules containing chromophores which may be quantified using their intrinsic absorption or fluorescence.
4. Dyes which can bind to protein noncovalently to generate a dye-protein complex resulting in a perturbation of the dye's absorption spectrum may be used to determine the presence or amount of protein in a sample. Some dyes require the presence of a metal ion, such as molybdenum, or tungsten in which case formation of a noncovalent complex of dye, metal ion, and protein results in the perturbation of the dye's absorption spectrum which may be used to determine the presence or amount of protein in a sample.
5. The competition of protein with a dye for coordination of Cu(II) results in an absorption signal that is proportional to the protein concentration(the Cu(II)/dye coordination complex has a different absorption spectrum from that of free dye, that is, dye which is not coordinated to Cu(II)).
U.S. Pat. No. 4,132,528 relates to assays for protein based on the biuret reaction. Dry assay elements of the '528 patent comprise a biuret reagent of CU(II) and a chelating agent therefore, e.g., CuSO.sub.4 and tartaric acid, or a complex of the two, and a buffer that provides a pH above about 12. When protein in an aqueous fluid, such as serum or cerebral spinal fluid (CSF), interacts with the biuret reagent at a pH above 12, a reaction between the cupric form of copper and the protein occurs to produce a violet color. The intensity of the color is directly proportional to the protein content of the serum, and the protein level can be measured by well known calorimetric analytical techniques. Unfortunately, the dry slide elements of the noted patent have lower sensitivity than desired, i.e., they cannot detect protein levels at or below about 200 mg/dL.
U.S. Pat. No. 4,786,605 describes dry analytical element formulations for quantification of protein having greater sensitivity than the elements of the '528 patent by replacing the biuret reagent(s) with a preformed Cu(II)/pyridylazo dye coordination complex (or free Cu(II) and free pyridylazo dye). When aqueous protein is added to the assay element cupric ion is displaced from the complex by the protein, and the absorption curve of the Cu(II)/dye complex is shifted to the absorption curve of the uncoordinated dye. Therefore, upon addition of protein, the absorption peak of the Cu(II)/dye complex is reduced in proportion to the amount of protein added, and from suitable calibration with samples having a known concentration of protein, an unknown concentration of protein in a liquid sample can be determined calorimetrically. The patent teaches that those elements have very good dynamic range and sensitivities as much as about 30 times greater than elements based on the biuret reaction.
The use of the '605 patent technology to quantify protein in urine samples, however, consistently shows that about 10 to 20% of the urine samples exhibit an unacceptable random positive bias compared with values obtained using a Coomassie Blue solution assay for protein concentration as the reference standard, i.e., the estimated protein concentration of certain patient urine samples (about 10 to 20% of the sample population), using the '605 patent technology, is greater than that determined using the Coomassie Blue-based assay method. It was deduced that this random positive bias was caused by the presence, in certain urine samples, of reducing agents such as ascorbic acid which cause the reduction of both Cu(II) and reducible groups on the dye such as nitro groups. A dry analytical element is desired which is not susceptible to the deficiencies of the aforementioned assay methodologies.
The reaction, in solution, of particular indicator dyes in complexation with protein and metal ions under acidic conditions which yields a measurable color change is well known. As mentioned above, an analytical assay method which works well in solution may not be readily adapted to dry analytical elements for reasons cited therein.
It is highly desirable that different proteins react equally with a dye to produce an optical signal which is related to the mass of protein present in the sample and independent of the nature of the protein. Urine contains variable quantities of bicarbonate (up to about 200 mM). High levels of bicarbonate in a urine specimen, if not diluted out or removed by sample pretreatment (such as by molecular size exclusion techniques), can introduce sufficient bicarbonate into the assay to produce very alkaline conditions which may render the assay unreliable or useless, since the dye-metal ion-protein interaction takes place at a low pH (1.5 to 3.5) and/or bicarbonate may interfere, additionally, through coordination of the metal ion. A need exists to provide dry analytical elements for the determination of protein which are not susceptible to the presence of reductants or bicarbonate, which produces a signal measurement that correlates substantially with the mass of protein present, that is, a signal measurement which is substantially (wherein substantially means suitable or acceptable for the specific protein measurement application, such as a measure of total protein in urine samples) independent of the nature of the protein, and can be used to quantitate protein in a range between approximately 5 to approximately 300 mg/dL and does not require predilution, preconcentration, or pretreatment of the sample to remove said interferents.
Unexpectedly, it has been found that dry analytical elements comprising indicator dyes in the presence of molybdate ion together with polymers comprising acrylamide, and hydroxycarboxylic acid compounds can be prepared which are suitable for use in is determining the amount or presence of protein in biological fluids.